Embodiments of the present invention relate to imaging, and more particularly to construction of an image with an enhanced depth of field.
Prevention, monitoring and treatment of physiological conditions such as cancer, infectious diseases and other disorders call for the timely diagnosis of these physiological conditions. Generally, a biological specimen from a patient is used for the analysis and identification of the disease. Microscopic analysis is a widely used technique in the analysis and evaluation of these samples. More specifically, the samples may be studied to detect presence of abnormal numbers or types of cells and/or organisms that may be indicative of a disease state. Automated microscopic analysis systems have been developed to facilitate speedy analysis of these samples and have the advantage of accuracy over manual analysis in which technicians may experience fatigue over time leading to inaccurate reading of the sample. Typically, samples on a slide are loaded onto a microscope. A lens or objective of the microscope may be focused onto a particular area of the sample. The sample is then scanned for one or more objects of interest. It may be noted that it is of paramount importance to properly focus the sample/objective to facilitate acquisition of images of high quality.
Digital optical microscopes are used to observe a wide variety of samples. A depth of field is defined as a measurement of a range of depth along a view axis corresponding to the in-focus portion of a three-dimensional (3D) scene being imaged to an image plane by a lens system. Images acquired via use of digital microscopes are typically acquired at high numerical apertures. The images obtained at the high numerical apertures are generally highly sensitive to a distance from a sample to an objective lens. Even a deviation of a few microns may be enough to throw a sample out of focus. Additionally, even within a single field of view of the microscope, it may not be possible to bring an entire sample into focus at one time merely by adjusting the optics.
Moreover, this problem is further exacerbated in the case of a scanning microscope, where the image to be acquired is synthesized from multiple fields of view. In addition to variations in the sample, the microscope slide has variations in its surface topography. The mechanism for translating the slide in a plane normal to the optical axis of the microscope may also introduce imperfections in image quality while raising, lowering and tiling the slide, thereby leading to imperfect focus in the acquired image. Additionally, the problem of imperfect focus is further aggravated in an event that a sample disposed on a slide is not substantially flat within a single field of view of the microscope. Specifically, these samples disposed on the slide may have significant amounts of material that is out of a plane of the slide.
A number of techniques have been developed for imaging that address problems associated with imaging a sample that has significant amounts of material out of plane. These techniques generally entail capturing entire fields of view of the microscope and stitching them together. However, use of these techniques results in inadequate focus when the depth of the sample varies significantly within a single field of view. Confocal microscopy has been employed to obtain depth information of a three-dimensional (3D) microscopic scene. However, these systems tend to be complex and expensive. Also, since confocal microscopy is typically limited to imaging of microscopic specimens, they are generally not practical for imaging macroscopic scenes.
Certain other techniques address the problem of automatic focusing when the depth of the sample varies significantly within a single field of view by acquiring and retaining images at multiple planes of focus. While these techniques provide images that are familiar to an operator of the microscope, these techniques require retention of 3-4 times the amount of data, and may well be cost-prohibitive for a high-throughput instrument.
In addition, certain other currently available techniques involve dividing an image into fixed areas and choosing the source image based on the contrast achieved in those areas. Unfortunately, use of these techniques introduces objectionable artifacts in the generated images. Moreover, these techniques tend to produce images of limited focus quality especially when confronted with samples disposed on a slide are not substantially flat within a single field of view, thereby limiting use of these microscopes in the pathology lab to diagnose abnormalities in such samples, particularly where the diagnosis requires high magnification (as with bone marrow aspirates).
It may therefore be desirable to develop a robust technique and system configured to construct an image with an enhanced depth of field that advantageously enhances image quality. Moreover, there is a need for a system that is configured to accurately image samples that have significant material out of a plane of the slide.